Nimble micromanagement of macromolecules

Intracellular protein homeostasis is managed by a complex series of enzymatic reactions, the terminal steps of which are catalyzed by dynamic protein aggregates called proteasomes, and more recently, immunoproteasomes.

The shear enormity of the numbers defies comprehension, dwarfing even the most aggressive congressional proposal to bail out the economy. Eighty to 90% of all intracellular protein is degraded through the ubiquitin-proteasome pathway. Of these proteins, the proteasome degrades about 500,000 different “slowly degraded protein” (or SDPs) substrates per minute, per cell.¹  That would be 3 × 10⁷ protein substrates per hour in a single cell. Interestingly, about 20% to 30% of all cellular protein synthesized in mammalian cells is degraded with a half-life of less than 10 minutes. These proteins are generally referred to as rapidly degraded polypeptides (or RDPs). A major fraction of RDPs includes defective ribosomal products (often referred to as DRiPs). DRiPS are defective proteins arising from the sometimes less than perfect process of translating genetic information from RNA into the language of protein.¹  RDPs generate another 1.3 × 10⁶ substrates degraded by the proteasome per minute, per cell. Collectively, the management of both SDPs and the RDPs generates over 2 million proteins to be degraded by the proteasome, per minute, per cell. This remarkable level of proteolytic activity produces more than 10⁸ oligopeptides per minute per cell. These oligopeptides are then metabolized by endopeptidiases and aminopeptidases to regenerate the pool of amino acids required for protein biogenesis, a process that transpires in only seconds.

The proteasome is a distinctive aggregate of globular proteins arranged in 4 symmetrical rings, each consisting of 7 α- and 7 β-subunits. The 4 rings form a barrellike structure with a central lumen. The barrel is flanked on top and on the bottom by the α-subunit ring while the 2 inner rings compose the β-subunit rings.²  These 4 rings are collectively referred to as the 20S proteasome. They house the proteases responsible for the catalytic functions of the proteasome and may, by itself, contribute to the degradation of nonubiquitylated substrates. The degradation of ubiquitylated substrates requires the addition of the 19S regulators to the 2 ends of the 20S proteasome, generating the 26S proteasome. The 19S regulator is also an aggregate of proteins that performs a number of vital functions. This regulator mediates the deubiquitylation of protein substrates, unfolds proteins from their complex tertiary structure into their primary structure, and then threads that unfolded, linearized protein into the central lumen of the 20S core.²  The core is where the protein meets its final fate, being degraded into smaller oligopeptides typically ranging in size from 3 to 23 amino acids. The process, as one might imagine, is fairly energy intensive, requiring the hydrolysis of ATP via ATPases integral to the 19S regulator.

The critical function of the proteasome, of course, is to degrade proteins back into the simpler building blocks from which they were assembled. This process is catalyzed through the function of discrete proteases embedded in the β-subunit rings. In fact, of the 14 distinct subunits that comprise the 20S proteasome (α-1, α2 … α7 and β1, β2 … β7), only 3 of these subunits contain true protease function, namely the β1, β2, and β5 subunits. These functional threonine proteases are unique among the vast numbers of different proteases found in the cytosol as the 20S proteases are processive in nature, catalyzing more than one cleavage site per substrate. With their active sites oriented inward toward the lumen of the 20S core, they cleave all recognized sites within a single substrate, generating enormous quantities of oligopeptides. These proteases are characterized by their distinctly different proteolytic functions (ie, different cleavage sites) and are referred to as the chymotrypsin-like (β1), trypsin-like (β2), and peptidylglutamyl-peptide hydrolyzing (PGPH; β5) activities, respectively. The latter of these enzymatic functions is also referred to as the postacidic or caspase-like activity.

Upon stimulation with interferon-γ (IFN-γ) or tumor necrosis factor-α (TNF-α), a change occurs in the protease subunit composition, leading to replacement of the β1, β2, and β5 subunits with new proteolytically distinct subunits called the LMP or βi-subunits. While each of these βi-subunits is genetically homologous to a specific constitutively expressed β-subunit, and can be readily inserted in place of the standard β1, β2, and β5 subunits, it possesses a distinctly different proteolytic function, cleaving proteins at different cleavage sites. Specifically, the β1i/LMP2 replaces the constitutively expressed β1-homolog, the β2i/LMP10 replaces the β2 homolog, and the β5i/LMP7 replaces the β5 homolog. In addition to the replacement of the β-subunits, there is a different regulatory subunit referred to as 11S or PA28.¹  The complete replacement of the β-subunits with the βi-subunits, and replacement of the 19S cap with the PA28 regulator now creates a unique structure often referred to as the immunoproteasome.

Immunoproteasomes are constitutively expressed in immune tissues and are expressed at much lower levels in other cell types. Immunoproteasomes play a major role in antigen presentation on MHC class I. These proteasomes, while of similar efficiency to the standard or constitutive proteasome, have different cleavage preferences so that the spectrum of antigenic peptides produced differs from that seen with the standard proteasome in other cells. These uniquely generated peptides influence CD8⁺ T-cell responses. While we are only now beginning to understand the role the immunoproteasome plays in normal cells, mounting evidence is beginning to suggest its dysregulation may play a role in diverse diseases including Huntingston disease, macular degeneration, and inflammatory bowel disease. With this biology in hand and the experience of bortezomib alongside them, Kuhn and colleagues have identified a series of immunoproteasome-specific inhibitors (IPSIs), using an in vitro screen.³  Using purified preparations of standard (or constitutive) proteasomes and immuno-proteasomes, they screened a rationally designed series of peptidyl-aldehydes, identifying one with unique specificity for the immunoproteasome. IPSI-001 preferentially targeted the β1i subunit, inducing accumulation of a host of varied proteins including ubiquitin-protein conjugates and proapoptotic proteins. Kuhn et al demonstrate that targeting the immunoproteasome in multiple myeloma cell lines and patient specimens with IPSI-001 produced a concentration dependent cytotoxicity. What is equally as interesting, and similar to the constitutive proteasome inhibitor experience, is that the IPSIs appear to overcome the acquired drug resistance phenotype in malignant cells.

Clearly, there is much to be learned about the differential functions of constitutive and immunoproteasomes. The demonstration that relatively unique inhibitors could be developed for one form of proteasome over another is remarkable and shows how a clearer understanding of even subtle differences in biology can lead to new hypotheses about treatment. While it will be years before we can declare this exercise a therapeutic success, it is abundantly clear that our ability to both understand and potentially manipulate the biology represents an exciting scientific advance.